Lewis-type sugar chain derivative

ABSTRACT

The object of the present invention is to provide a new SLe-type sugar chain derivative represented by the following formula (I), which is useful as a pharmaceutical and which contains moranoline: ##STR1## wherein R 1  represents hydrogen, a lower alkyl, a lower alkenyl or a lower alkynyl; R 2  and R 3  differ from each other and represent a galactosyl, sialylgalactosyl or fucosyl group; R 4  represents a hydroxyl group or an acetamido group. The present invention comprises a sugar chain derivative containing a moranoline, and having a new structure. Possessing cell adhesion inhibitory activity, the compounds of the present invention antagonizes selectin, serving well in the treatment of inflammation, inflammation-associated thrombosis, asthma and rheumatism, and in the prevention and treatment of immunological diseases and cancer.

TECHNICAL FIELD

The present invention relates to a new sialyl Lewis X (hereinafterabbreviated SLe^(x)) type and sialyl Lewis A (hereinafter abbreviatedSLe^(a)) type ganglioside sugar chain derivatives represented by thefollowing formula (I), which are useful in pharmaceutical fields such astreatment and prevention of inflammation, intimation-associatedthrombosis, asthma, rheumatism, immunological diseases and cancer:##STR2## wherein R¹ represents hydrogen, a lower alkyl, a lower alkenylor a lower alkynyl; R² and R³ independently represent galactosyl,sialylgalactosyl or fucosyl; R⁴ represents a hydroxyl or acetamido.

BACKGROUND ART

Recent studies have demonstrated that the sugar chains of glycolipidsand glycoproteins function as receptors of hormones, bacterial toxins,viruses and others and are profoundly involved in basic dynamicbiological phenomena such as cell recognition,differentiation/proliferation, adhesion, transformation, immunity andaging.

Cell surface sugar chains, closely associated with blood groupsubstances such as those of the ABO (H antigen), Lewis (Le antigen) andIi blood types, are also detected as cancer-related sugar chainantigens. For this reason, there have been studies to apply monoclonalantibodies, that specifically recognize this series of sugar chains, tocancer diagnosis and treatment.

The sialyl Le type sugar chain antigen, a tetrasaccharide sugar chainantigen basically composed of α(2→3) sialyl and α(1→3 and 1→4)fucosylated lactosamine, and found in sialoglycoprotein sugar chains aswell as in gangliosides, has been used in serologic diagnosis of canceras a sugar chain antigen for pulmonary adenocarcinoma and digestivetract cancer. Also, sialyl Le^(x) sugar chains have very recently beenreported as functioning as sugar chain ligands for the leukocyteadhesion factor appearing on vascular endothelial cells duringintimation [Lowe, J. B., et al., Cell, 63, 475-484 (1990)].

It is very difficult, however, to obtain these sugar chains as a puresingle compound from the living body because they are present in thecell surface layer only in trace amounts.

Against this background various synthetic compounds, includingderivatives, have been studied, but none have been known to havemoranoline (1-deoxynojirimycin) as a sugar chain component like thecompounds of the present invention.

The object of the present invention is to provide a sugar chainderivative that is useful as a pharmaceutical and has a new structure.

DISCLOSURE OF INVENTION

Through extensive research, the present inventors found that the objectof the present invention can be accomplished by four types of compoundsrepresented by general formula (I): i. SLe^(x) -type sugar chainderivatives, ii. Le^(x) -type sugar chain derivatives, iii. SLe^(a)-type sugar chain derivatives, which are isomers of the derivatives ofterms i. and ii. and have a very similar three-dimensional structure,and iv. Le^(a) -type sugar chain derivatives. The inventors made furtherinvestigations based on this finding, and developed the presentinvention.

The essence of the present invention exists in the structure of thecompounds of represented by general formula (I). The compounds of theinvention are new compounds not described in literatures, and possessexcellent pharmacologic action as stated later.

The lower alkyl for R¹ in the formula (I), is preferably a linear orbranched alkyl having 1 to 7 carbon atoms, such as methyl, ethyl,n-propyl, isopropyl, n-butyl, isobutyl, t-butyl, n-pentyl, isopentyl,n-hexyl, isohexyl, n-heptyl or isoheptyl.

The lower alkenyl for R¹ is preferably a linear or branched alkenylhaving i to 7 carbon atoms, such as vinyl, propenyl, butenyl, pentenyl,hexenyl or heptenyl.

The lower alkynyl for R¹ is preferably a linear or branched alkynylhaving 1 to 7 carbon atoms, such as ethynyl, propynyl, butynyl,pentynyl, hexynyl or heptynyl.

The compounds of the present invention are exemplified by the followingcompounds as well as the compounds described in the Examples ofpreparation. Those exemplified compounds, however, only show a part ofthe present invention. Accordingly, the compounds of the presentinvention are not limited to those exemplified compounds.

O-(5-acetamido-3,5-dideoxy-D-glycero-α-D-galacto-2-nonulopyranosylomcacid)-(2→3)-O-(β-D-galactopyranosyl)-(1→4)-O-(α-L-fucopyranosyl)-(1→3)]-N-meth-1,5-dideoxy-1,5-imino-D-glucitol

O-(5-acetamido-3,5-dideoxy-D-glycero-α-D-galacto-2-nonulopyranosylonicacid)-(2→3)-O-(β-D-galactopyranosyl)-(1→4)-O-[(α-L-fucopyranosyl)-(1→3)]-N-methyl-1,5-dideoxy-1,5-imino-D-glucitol

O-(5-acetamido-3,5-dideoxy-D-glycero-α-D-galacto-2-nonulopyranosylonicacid)-(2→3)-O-(β-D-galactopyranosyl)-(1→4)-O-[(α-L-fucopyranosyl)-(1→3)]-N-methyl-1,5-dideoxy-1,5-imino-D-glucitol

O-(5-acetamido-3,5-dideoxy-D-glycero-α-D-galacto-2-nonulopyranosylomcacid)-(2→3)-O-(β-D-galactopyranosyl)-(1→4)-O-[(α-L-fucopyranosyl)-(1→3)]-N-vinyl-1,5-dideoxy-1,5-imino-D-glucitol

O-(5-acetamido-3,5-dideoxy-D-glycero-α-D-galacto-2-nonulopyranosylomcacid)-(2→3)-O-(β-D-galactopyranosyl)-(1→4)-O-[(α-L-fucopyranosyl)-(1→3)]-N-allyl-1,5-dideoxy-1,5-imino-D-glucitol

O-(5-acetamido-3,5-dideoxy-D-glycero-α-D-galacto-2-nonulopyranosylomcacid)-(2→3)-O-(β-D-galactopyranosyl)-(1→4)-O-[(α-L-fucopyranosyl)-(1→3)]-2-acetamido-1,2,5-trideoxy-N-propyl-1,5-imino-D-glucitol

O-(5-acetamido-3,5-dideoxy-D-glycero-α-D-galacto-2-nonulopyranosylomcacid)-(2→3)-O-(β-D-galactopyranosyl)-(1→4)-O-[(α-L-fucopyranosyl)-(1→3)]-2-acetamido-1,2,5-trideoxy-N-pentyl-1,5-imino-D-glucitol

O-(5-acetamido-3,5-dideoxy-D-glycero-α-D-galacto-2-nonulopyranosylonicacid)-(2→3)-O-(β-D-galactopyranosyl)-(1→4)-O-[(α-L-fucopyranosyl)-(1→3)]-2-acetamido-1,2,5-trideoxy-N-heptyl-1,5-imino-D-glucitol

O-(5-acetamido-3,5-dideoxy-D-glycero-α-D-galacto-2-nonulopyranosylonicacid)-(2→3)-O-(β-D-galactopyranosyl)-(1→3)-O-[(α-L-fucopyranosyl)-(1→4)]-N-methyl-1,5-dideoxy-1,5-imino-D-glucitol

O-(5-acetamido-3,5-dideoxy-D-glycero-α-D-galacto-2-nonulopyranosylonicacid)-(2→3)-O-(β-D-galactopyranosyl)-(1→3)-O-[(α-L-fucopyranosyl)-(1→4)]-N-propyl-1,5-dideoxy-1,5-imino-D-glucitol

O-(5-acetamido-3,5-dideoxy-D-glycero-α-D-galacto-2-nonulopyranosylonicacid)-(2→3)-O-(β-D-galactopyranosyl)-(1→3)-O-[(α-L-fucopyranosyl)-(1→4)]-N-pentyl-1,5-dideoxy-1,5-imino-D-glucitol

O-(5-acetamido-3,5-dideoxy-D-glycero-α-D-galacto-2-nonulopyranosylonicacid)-(2→3)-O-(β-D-galactopyranosyl)-(1→3)-O-[(α-L-fucopyranosyl)-(1→4)]-N-vinyl-1,5-dideoxy-1,5-imino-D-glucitol

O-(5-acetamido-3,5-dideoxy-D-glycero-α-D-galacto-2-nonulopyranosylonicacid)-(2→3)-O-(β-D-galactopyranosyl)-(1→3)-O-[(α-L-fucopyranosyl)-(1→4)]-N-allyl-1,5-dideoxy-1,5-imino-D-glucitol

O-(5-acetamido-3,5-dideoxy-D-glycero-α-D-galacto-2-nonulopyranosylonicacid)-(2→3)-O-(β-D-galactopyranosyl)-(1→3)-O-[(α-L-fucopyranosyl)-(1→4)]-2-acetamido-1,2,5-trideoxy-N-propyl-1,5-imino-D-glucitol

O-(5-acetamido-3,5-dideoxy-D-glycero-α-D-galacto-2-nonulopyranosylonicacid)-(2→3)-O-(β-D-galactopyranosyl)-(1→3)-O-[(α-L-fucopyranosyl)-(1→4)]-2-acetamido-1,2,5-trideoxy-N-pentyl-1,5-imino-D-glucitol

O-(5-acetamido-3,5-dideoxy-D-glycero-α-D-galacto-2-nonulopyranosylonicacid)-(2→3)-O-(β-D-galactopyranosyl)-(1→3)-O-[(α-L-fucopyranosyl)-(1→4)]-2-acetamido-1,2,5-trideoxy-N-heptyl-1,5-imino-D-glucitol

The compounds of the present invention possess cell adhesion inhibitoryactivity, as described in detail in Test Examples later.

Cell adhesion occurs as follows: for example, when leukocytes permeatethrough an inflammatory area, they adhere to vascular endotheliumthrough capillary blood flow (first adhesion) and roll on theendothelial surface. Next, the leukocytes strongly adhere to theendothelial surface (second adhesion), then migrate into theinflammatory tissue through endothelia intercellular spaces. Thus, celladhesion occurs in two stages until leukocytes migrate into inflammatorytissue.

As such, cell adhesion is known to be mediated by a group of proteinscalled as selectin, present in endothelial cells, and its ligand, or asugar chain of a ganglioside having an Le-type sugar chain antigen or asialyl Le-type sugar chain antigen, present on the leukocyte surface.

Also, the sialyl Le-type sugar chain antigen, which has a sialyl group,is 30 times as potent as the Le-type sugar chain antigen, which has nosialyl group, in adhesion activity [Proc. Natl. Acad. Sci. USA, 88(1991)].

Selectin appears on endothelial cells upon endothelial cell stimulationwith interleukin 1 or TNF and adheres to leukocytes, but there are sometypes of selectin which are thought to form thrombi upon the appearence.

On the other hand, the sialyl Le-type or Le-type sugar chain antigen isthought to be associated with cancer metastasis, since it also exists inmetastatic cancer cells. The sialyl Le^(a) sugar chain antigen, inparticular, appears on the surface of almost all types of cancer cells,mainly digestive tract cancer and is thought to play a key role invascular invasion and metastasis of cancer cells.

Any substance having cell adhesion inhibitory activities inhibitsendothelin cell from adhering on leukocytes and cancer cells, and ishence useful in treating and preventing inflammation,inflammation-associated thrombosis, rheumatism, asthma, infectiousdiseases, immunological diseases, AIDS and cancer.

The compounds of the present invention are therefore useful as ananti-inflammatory agent, antithrombotic therapeutic agent,antiasthmatic/rheumatic therapeutic agent, anti-infectious therapeuticagent, anti-ADS therapeutic agent, and therapeutic and prophylacticagent for immunological diseases and cancer.

The Le^(x) derivative represented by formula (I) mentioned above can,for example, be prepared as follows:

After the N-tert-butoxycarbonyl derivative of 1-deoxynojirimycinrepresented by the following formula (wherein Ac and Boc represent anacetyl group and a tertbutoxycarbonyl group, respectively; the sameapplies below) ##STR3## is converted into the followingN-benzyloxycarbonyl derivative (wherein Z represents a benzyloxycarbonylgroup; the same applies below) ##STR4## the 4- and 6-hydroxyl groups areprotected with a benzylidene group to derive a compound of the followingformula (wherein Ph represents a phenyl group; the same applies below):##STR5##

This reaction is followed by position-selective chloroacetylation andsubsequent acetylation of the 2-hydroxyl group and dechloroacetylation,to yield an acetyl derivative represented by the following formula:##STR6##

This acetyl derivative is condensed in an inert solvent with thefollowing compound, methyl-2,3,4-tri-O-benzyl-1-thio-β-L-fucopyranoside##STR7## (hereinafter referred to as compound A) (wherein Bn and Merepresent a benzyl group and a methyl group, respectively; the sameapplies below), previously synthesized from L-fucose via several steps,to yield an α-(1→3)-glycoside represented by the following formula athigh yield: ##STR8##

This reaction is carded out in an aprofic solvent in the presence ofdimethyl(methylthio)sulfonium triflate (DMTST) or N-iodosuccinimide(NIS) within the temperature range of 0° C.-50° C. for about 1 to 4hours. The condensation reaction may be preceded by dehydration using adesiccant such as Molecular Sieves if necessary.

Subsequently, the benzylidene group is reductively split off to yield acompound represented by the following formula. This reaction is carriedout within the temperature range of 0° C.-50° C. for about 30 minutes to2 hours: ##STR9##

Condensation of the above compound withmethyl-2,3,4,6-tetra-O-benzoyl-1-thio-β-D-galactopyranoside (wherein Bzrepresents a benzoyl group; the same applies below) ##STR10## in aninert solvent results in formation of the corresponding trisaccharidederivative: ##STR11##

This reaction is carded out within the temperature range of 0° C.-50° C.for about one to several hours. The above-mentioned basic or acidiccatalysts can be used as condensing agents; it is preferable that thecondensation reaction be carded out in the presence of N-iodosuccinimide(NIS) and trifluoromethanesulfonic acid. The condensation reaction maybe preceded by dehydration using a desiccant as necessary. Next, thebenzyl group and the benzyloxycarbonyl group are removed by catalytichydrogenation to yield the following compound, ##STR12## which is thenreacted with an alkoxide in an alcohol solvent to remove all O-acyl(acetyl and benzoyl) groups, to synthesize an Le^(x) sugar chainrepresented by the following formula, that contains 1-deoxynojirimycin.This reaction is carried out within the temperature range of 0° C.-50°C. for 1 to 2 days. ##STR13##

On the other hand, the following compound ##STR14## is condensed withthe methylthioglycoside derivative of sialyl-α-(2→3)-galactoserepresented by the following formula (hereinafter referred to ascompound B) ##STR15## in an inert solvent in the presence of acondensing agent to yield the corresponding tetrasaccharide sugar chain:##STR16##

This reaction is carded out within the temperature range of 0° C.-50° C.for about 1 to 2 days. The above-mentioned basic or acidic catalysts canbe used as condensing agents. It is preferable that the condensationreaction be carried out in the presence of N-iodosuccinimide (NIS) andtrifluoromethanesulfonic acid. The condensation reaction may be precededby dehydration using a desiccant if necessary.

Next, the benzyl group and the benzyloxycarbonyl group are removed bycatalytic hydrogenation to yield the following compound, ##STR17## whichis then reacted with an alkoxide in an alcohol solvent to remove allO-acyl (acetyl and benzoyl) groups, followed by hydrolysis of carboxylicacid methyl ester with an aqueous alkali solution and neutralization, toyield the desired compound, a sialyl Le^(x) sugar chain containing1-deoxynojirimycin: ##STR18##

This reaction is carded out within the temperature range of 0° C.-50° C.for 1 to 2 days. Although it is preferable that the alkoxide be sodiummethoxide and the alkali in aqueous solution be potassium hydroxide,these are not to be construed as limitative.

BEST MODES FOR CARRYING OUT THE INVENTION EXAMPLES

The present invention is hereinafter illustrated in more detail by meansof the following working examples and test example. Optical rotation wasmeasured at 25° C. without exception.

[EXAMPLE 1]

Preparation of Le^(x) and sialyl Le^(x) -type sugar chain derivatives

(1) Preparation of2,3,4,6-tetra-O-acetyl-N-tert-butoxycarbonyl-1,5-dideoxy-1,5-imino-D-glucitol:##STR19##

N-(tert-butoxycarbonyl)- 1,5-dideoxy- 1,5-imino-D-glucitol (19.36 g) wasdissolved in pyridine (100 ml). To this solution, acetic anhydride (50ml) was added, followed by overnight stirring at room temperature. Aftercompletion of the reaction, methanol (50 ml) was added at 0° C.,followed by concentration under reduced pressure. The resulting residuewas extracted with dichloromethane and washed with 2N hydrochloric acidand water. After drying over sodium sulfate, the extract layer wasfiltered and thoroughly washed with dichloromethane. The filtrate wascombined with washings, followed by concentration under reducedpressure. The resulting residue was subjected to column chromatography(Wako Gel C-200, dichloromethane) to yield compound (1) (31.70 g,quantitative).

Physical property data 1. Optical rotation: [α]_(D) +0.71 (C=0.842,dichloromethane) 2. Elemental analysis (for C₁₉ H₂₉ NO₁₀) Calculated.:C, 52.90%; H, 6.78%; N, 3.25% Found: C, 53.0%; H, 7.00%; N, 3.42%

(2) Preparation of2,3,4,6-tetra-O-acetyl-N-benzyloxycarbonyl-1,5-dideoxy-1,5-imino-D-glucitol

Compound (1) (21.04 g) was dissolved in dichloromethane (30 ml). To thissolution, trifluoroacetic acid (18.79 ml, 5 equivalents) was added,followed by overnight stirring at room temperature. After completion ofthe reaction, the reaction mixture was concentrated under reducedpressure at 45° C. Ether was added to the resulting residue, followed byseveral decantations. The obtained solid was dissolved in methanol (30ml). The solution was neutralized with ion exchange resin AmberliteIR-410 (OH⁻). The resin was filtered out and thoroughly washed withmethanol. The filtrate was combined with washings, followed byconcentration under reduced pressure. The resulting residue wasdissolved in dichloromethane (40 ml) and pyridine (15 ml). To thissolution, benzyl chloroformate (22.96 g, 3 equivalents) was added at 0°C., followed by overnight stirring at water temperature. Aftercompletion of the reaction, methanol was added thereto, followed byconcentration under reduced pressure at 45° C. The residue was extractedwith dichloromethane and washed with 2N hydrochloric acid and water. Theextract layer was dried over sodium sulfate, filtered, and washed withdichloromethane. The filtrate was combined with washings, followed byconcentration under reduced pressure. The resulting residue wassubjected to column chromatography (Wako Gel C-200, dichloromethane) toyield compound (2) (21.53 g, 92.0%).

Physical property data 1. Optical rotation: [α]_(D) - 6.06° (C=0.990,dichloromethane) 2. Elemental analysis (for C₂₂ H₂₇ NO₁₀) Calculated: C,56.77%; H, 5.85%; N, 3.01% Found: C, 57.03%; H, 5.87%; N, 3.09%

(3) Preparation of N-benzyloxycarbonyl-1,5-dideoxy-1,5-imino-D-glucitol

Compound (2) (9.13 g) was dissolved in methanol (100 ml). To thissolution, sodium methoxide was added at 0° C. until the pH became almost12, followed by stirring at 0° C. for 1 hour. After completion of thereaction, the solution was neutralized with ion exchange resin AmberliteIR-120 (H⁺). The resin was filtered out and washed with methanol. Thefiltrate was combined with washings, followed by concentration underreduced pressure. The resulting residue was subjected to columnchromatography (Wako Gel C-200, dichloromethane/methanol=50/1 ) to yieldcompound (3) (4.96 g, 85.1%).

Physical property data 1. Optical rotation: [α]_(D) -13.58° (C=0.692,methanol) 2. Elemental analysis (for C₁₄ H₁₉ NO₆) Calculated: C, 56.56%;H, 6.44%; N, 4.71% Found: C, 56.60%; H, 6.39%; N, 4.96%

(4) Preparation of4,6-O-benzylidene-N-benzyloxycarbonyl-1,5-dideoxy-1,5-imino-D-glucitol:##STR20##

Compound (3) (9.21 g) was dissolved in N,N-dimethylformamide (27 ml). Tothis solution, Drierite (10 g), a desiccant, was added, followed bystirring at room temperature for 1 hour. Then, benzaldehydedimethylacetal (13.94 ml, 3 equivalents) and a catalytic amount ofp-toluenesulfonic acid were added, followed by overnight stirring atroom temperature. After completion of the reaction, methanol was addedthereto, and the mixture was neutralized with ion exchange resinAmberlite IR-410 (OH⁻). The resin and Drieritc were filtered off,followed by washing with methanol.

The filtrate was combined with washings, followed by concentration underreduced pressure. The resulting residue was subjected to columnchromatography (Wako Gel C-200, ethyl acetate/hexane=1/2) to yieldcompound (4) (8.33 g, 69.8%), which was then crystallized from ethylacetate/hexane to yield a white crystal.

Physical property data 1. Optical rotation: [α]_(D) +8.69° (C=0.690,dichloromethane) 2. Elemental analysis (for C₂₁ H₂₃ NO₆) Calculated: C,65.44%; H, 6.02%; N, 3.63% Found: C, 65.53%; H, 5.72%; N, 3.42%

(5) Preparation of4,6-O-benzylidene-N-benzyloxycarbonyl-3-O-chloroacetyl-1,5-dideoxy-1,5-imino-D-glucitol

Compound (4) (1 g) was dissolved in dichloromethane (80 ml). To thissolution, lutidine (0.6 ml, 2 equivalents) was added. After thissolution was cooled to -20° C., a solution of chloroacetyl chloride(0.25 ml, 1.2 equivalents) in dichloromethane (20 ml) was added drop bydrop. The reaction mixture was stirred at -20° C. for 1.5 hours. Aftercompletion of the reaction was confirmed by TLC, the mixture wasextracted with dichloromethane and washed with 2N hydrochloric acid andwater. The extract layer was dried over sodium sulfate and faltered. Thefiltrate was combined with washings, followed by concentration underreduced pressure. The resulting residue was subjected to columnchromatography (Wako Gel C-200, eluents: a) dichloromethane, b) 500:1dichloromethane:methanol) to yield compound (5) (0.81 g, 68%) fromeluate b).

Physical property data 1. Optical rotation: [α]_(D) -5.15° (C=0.582,dichloromethane) 2. Elemental analysis (for C₂₃ H₂₄ NO₇ Cl) Calculated:C, 59.81%; H, 5.24%; N, 3.03% Found: C, 60.03%; H, 4.98%; N, 3.29%

(6) Preparation of2-O-acetyl-4,6-O-benzylidene-N-benzyloxycarbonyl-3-O-chloroacetyl-1,5odideoxy-1,5-imino-D-glucitol

Compound (5) (410 mg) was dissolved in dichloromethane (4 ml) andpyridine (2 ml). After this solution was cooled to -20° C., acetylchloride (0.1 ml, 1.5 equivalents) was added, followed by stirring at-20° C. to 0° C. for 4.5 hours. After completion of the reaction wasconfirmed by TLC, the reaction mixture was extracted withdichloromethane and washed with 2N hydrochloric acid and water. Theextract layer was dried over sodium sulfate and filtered. The filtratewas combined with washings, followed by concentration under reducedpressure at 20° C., to yield compound (6) (450 mg, quantitative).

Physical property data 1. Optical rotation: [α]_(D) -19.48° (C=1.016,dichloromethane) 2. Elemental analysis (for C₂₅ H₂₆ NO₈ Cl) Calculated:C, 59.59%; H, 5.20%; N, 2.78% Found: C, 59.69%; H, 5.14%; N, 3.05%

(7) Preparation of2-O-acetyl-4,6-O-benzylidene-N-benzyloxycarbonyl-1,5-dideoxy-1,5-imino-D-glucitol

Compound (6) (250 mg) was dissolved in pyridine (10 ml). To thissolution, water (2 ml) was added, followed by overnight stirring at roomtemperature. After completion of the reaction was confirmed by TLC, thereaction mixture was extracted with dichloromethane and washed with 2Nhydrochloric acid and water. The extract layer was dried over sodiumsulfate and filtered. The filtrate was combined with washings, followedby concentration under reduced pressure. The resulting residue wassubjected to column chromatography (Wako Gel C-200, eluent: 500:1dichloromethane:methanol) to yield compound (7) (210 mg, quantitative).

Physical property data 1. Optical rotation: [α]_(D) -12.47° (C=0.930,dichloromethane) 2. Elemental analysis (for C₂₃ H₂₅ NO₇) Calculated: C,64.63%; H, 5.90%; N, 3.28% Found: C, 64.72%; H, 5.70%; N, 3.38%

(8) Preparation ofO-(2,3,4-tri-O-benzyl-α-L-fucopyranosyl)-(1→3)-2-O-acetyl-4,6-O-benzylidene-N-benzyloxycarbonyl-1,5-dideoxy-1,5-imino-D-glucitol:##STR21##

Compound (7) (30 mg) andmethyl-2,3,4-tri-O-benzyl-1-thio-β-L-fucopyranoside (39 mg, 1.2equivalents) were dissolved in benzene (16 ml). To this solution,Molecular Sieves 4A (100 mg), a desiccant, was added, followed byovernight stirring at room temperature. Next,dimethyl(methylthio)sulfonium triflate (97 mg, 4 equivalents) was addedat 7° C., followed by stirring at 7° C. to room temperature for 2.5hours. After completion of the reaction was confirmed by TLC, methanol(2 ml) and triethylamine (412) ml) were added at 0° C., followed byseparation of the Molecular Sieves by filtration. Then, the filtrate wascombined with washings, followed by concentration under reducedpressure. The resulting residue was extracted with dichloromethane aridwashed with sodium hydrogen carbonate and water. The extract layer wasdried over sodium sulfate, faltered, and concentrated under reducedpressure. The resulting residue was subjected to column chromatography(Wako Gel C-200, eluent: 1:4 ethyl acetate:hexane) to yield compound (8)(55 mg, 92%).

Physical property data 1. Optical rotation: [α]_(D) -93.64° (C=1.023,dichloromethane) 2. Elemental analysis (for C₅₀ H₅₃ NO₁₁) Calculated: C,71.16%; H, 6.33%; N, 1.66% Found: C, 71.33%; H, 6.54%; N, 1.69%

(9) Preparation ofO-(2,3,4-tri-O-benzyl-α-L-fucopyranosyl)-(1→3)-2-O-acetyl-6-O-benzyl-N-benzyloxycarbonyl-1,5-dideoxy- 1,5-imino-D-glucitol

Compound (8) (1 g) was dissolved in tetrahydrofuran (30 ml). To thissolution, Molecular Sieves 3A (2 g), a desiccant, was added, followed bystirring at room temperature for 2 hours. Next, sodium cyanoborohydride(1.2 g, 15 equivalents <) was added for activation, after whichhydrochloric acid/ether was added thereto drop by drop until generationof gaseous hydrogen from the reaction mixture stopped, followed bystirring at room temperature for 30 minutes. After completion of thereaction was confirmed by TLC, triethylamine was added thereto at 0° C.to neutralize the reaction mixture, followed by filtration. TheMolecular Sieves was thoroughly washed with dichloromethane andmethanol. The filtrate was combined with washings, followed byconcentration under reduced pressure. The resulting residue wasextracted with dichloromethane and washed with water. The extract layerwas dried over sodium sulfate, filtered, and concentrated under reducedpressure. The resulting residue was subjected to column chromatography(Wako Gel C-200, eluent: 1:2 ethyl acetate:hexane) to yield compound (9)(810 mg, 81%).

Physical property data 1. Optical rotation: [α]_(D) -50.73° (C=0.820,dichloromethane) 2. Elemental analysis (for C₅₀ H₅₅ NO₁₁) Calculated: C,70.99%; H, 6.55%; N, 1.66% Found: C, 71.06%; H, 6.81%; N, 1.70%

(10) Preparation ofO-(2,3,4,6-tetra-O-benzoyl-β-D-galactopyranosyl)-(1→4)-O-[(2,3,4-tri-O-benzyl-α-L-fucopyranosyl)-(1→3)]-2-O-acetyl-6-O-benzyl-N-benzyloxycarbonyl-1,5-dideoxy-1,5-imino-D-glucitol:##STR22##

Compound (9) (130 mg)andmethyl-2,3,4,6-tetra-O-benzoyl-1-thio-β-D-galactopyranoside (222 mg, 2equivalents) were dissolved in dichloromethane (12 ml). To thissolution, Molecular Sieves 4A (400 mg), a desiccant, was added, followedby overnight stirring at room temperature. After cooling the mixture to0° C., N-iodosuccinimide (160 mg, 4 equivalents) andtrifluoromethanesulfonic acid (6 μl, 0.4 equivalents) were addedthereto, followed by overnight stirring at 0° C. to room temperature.After completion of the reaction was confirmed by TLC, the mixture wasfiltered to separate the Molecular Sieves, then extracted withdichloromethane. The extract was washed with sodium carbonate, sodiumthiosulfate and water. The extract layer was dried over sodium sulfateand filtered. The filtrate was combined with washings and concentratedunder reduced pressure. The resulting residue was subjected to columnchromatography (Merck Kiesel Gel 60, eluent: 1:2 ethyl acetate:hexane)to yield compound (10) (150 mg, 69%).

Physical property data 1. Optical rotation: [α]_(D) -10.40° (C=1.172,dichloromethane) 2. Elemental analysis (for C₈₄ H₈₁ NO₂₀) Calculated: C,70.83%; H, 5.73%; N, 0.98% Found: C, 70.68%; H, 5.90%; N, 0.80%

(11) Preparation ofO-(2,3,4,6-tetra-O-benzoyl-β-D-galactopyranosyl)-(1→4)-O-[(.alpha.-L-fucopyranosyl)-1→3)]-2-O-acetyl-1,5-dideoxy-1,5-imino-D-glucitol

Compound (10) (30 mg) was dissolved in methanol (3 ml) and formic acid(3 ml). To this solution, palladium black (30 mg), previously activated,was added, followed by catalytic hydrogenation at room temperature for 3days. After completion of the reaction was confirmed by TLC, thecatalyst was faltered out and washed with methanol. The filtrate wascombined with washings, followed by concentration under reducedpressure. The resulting residue was subjected to column chromatography(Wako Gel C-200, eluent: 30:1 dichloromethane:methanol) to yieldcompound (11) (19 mg, quantitative).

Physical property data 1. Optical rotation: [α]_(D) -7.58° (C=0.343,methanol) 2. Elemental analysis (for C₄₈ H₅₁ NO₁₈) Calculated: C,62.00%; H, 5.53%; N, 1.51% Found: C, 61.97%; H, 5.23%; N, 1.43%

(12) Preparation ofO-(β-D-galactopyranosyl)-(1→4)-O-[(α-L-fucopyranosyl)-(1→3)]-1,5-dideoxy-1,5-imino-D-glucitol

Compound (11 ) (24 mg) was dissolved in methanol (10 ml). To thissolution, sodium methoxide was added until the pH became almost 12,followed by overnight stirring at room temperature. After completion ofthe reaction was confirmed by TLC, the solution was neutralized with ionexchange resin Amberlite IR-120 (H⁺). The resin was filtered out andwashed with methanol. The filtrate was combined with washings, followedby concentration under reduced pressure. The resulting residue wassubjected to gel filtration (Sephadex LH-20, eluent: 3:1 ethanol:water)to yield compound (12) (16 mg, quantitative).

Physical property data 1. Optical rotation: [α]_(D) -23.98° (C=0.467,water:ethanol=2:1 ) 2. Elemental analysis (for C₁₈ H₃₃ NO₁₃) Calculated:C, 45.86%; H, 7.06%; N, 2.97% Found: C, 46.01%; H, 7.17%; N, 2.97%

(13) Preparation of O-(methyl5-acetamido-4,7,8,9-tetra-O-acetyl-3,5-dideoxy-D-glycero-α-D-galacto-2-nonulopyranosylonate)-(2→3)-O-(2,4,6-tri-O-benzoyl-β-D-galactopyranosyl)-(1→4)-O-[(2,3,4-tri-O-benzyl-α-L-fucopyranosyl)-(1→3)]-2-O-acetyl-6-O-benzyl-N-benzyloxycarbonyl-1,5-dideoxy-1,5-imino-D-glucitol:##STR23##

Compound (9) (70 mg) and compound B (124 mg, 1.5 equivalents) weredissolved in dichloromethane (10 ml). To this solution, Molecular Sieves4A (250 mg), a desiccant, was added, followed by overnight stirring atroom temperature. After coloring the mixture to 0° C., N-iodosuccinimide(56 mg, 3 equivalents) and trifluoromethanesulfonic acid (2.2 μl, 0.3equivalents ) were added, followed by overnight stirring at 0° C. toroom temperature. After completion of the reaction was confirmed by TLC,the Molecular Sieves was filtered out and then the filtrate wasextracted with dichloromethane and washed with sodium carbonate, sodiumthiosulfate and water. The extract layer was dried over sodium sulfateand filtered. The filtrate was combined with washings and concentratedunder reduced pressure. The resulting residue was subjected to columnchromatography (Wako Gel C-300, eluent: 3:2 ethyl acetate:hexane) toyield compound (13) (90 mg, 61%).

Physical property data 1. Optical rotation: [α]_(D) -10.69° (C=0.673,dichloromethane) 2. Elemental analysis (for C₉₇ H₁₀₄ N₂ O₃₁) Calculated:C, 64.95%; H, 5.84%; N, 1.56% Found: C, 64.75%; H, 5.56%; N, 1.58%

(14) Preparation of O-(methyl5-acetamido-4,7,8,9-tetra-O-acetyl-3,5-dideoxy-D-glycero-α-D-galacto-2-nonulopyranosylonate)-(2→3)-O-(2,4,6-tri-O-benzoyl-β-D-galatcopyranosyl)-(1→4)-O-[(α-L-fucopyranosyl)-(1→3)]-2-O-acetyl-1,5-dideoxy-1,5-imino-D-glucitol

Compound (13) (70 mg) was dissolved in methanol (6 ml) and acetic acid(6 ml). To this solution, palladium black (70 mg), previously activated,was added, followed by catalytic hydrogenation at room temperature for 3days. After completion of the reaction was confirmed by TLC, thepalladium was filtered out and washed with methanol. The filtrate wascombined with washings, followed by concentration under reducedpressure. The resulting residue was subjected to column chromatography(Wako Gel C-200, eluent: 25:1 dichloromethane:methanol) to yieldcompound (14) (50 mg, quantitative).

Physical property data 1. Optical rotation: [α]_(D) -20.25° (C=0.800,methanol) 2. Elemental analysis (for C₆₁ H₇₄ N₂ O₂₉) Calculated: C,56.39%; H, 5.74%; N, 2.16% Found: C, 56.48%; H, 5.61%; N, 2.15%

(15) Preparation ofO-(5-acetamido-3,5-dideoxy-D-glycero-α-D-galacto-2-nonulopyranosylonicacid)-(2→3)-O-([3-D-galactopyranosyl)-(1→4)-O-[(α-L-fucopyranosyl)-(1→3)]-1,5-dideoxy-1,5-imino-D-glucitol:##STR24##

Compound (14) (50 mg) was dissolved in methanol (6 ml). To thissolution, sodium methoxide was added until the pH became almost 12,followed by overnight stirring at room temperature. Then, a 0.2N aqueoussolution of potassium hydroxide (2 ml) was added, followed by stirringat room temperature for 2 days. After completion of the reaction wasconfirmed by TLC, the mixture was neutralized with ion exchange resinAmberlite IR-120 (H⁺). The resin was filtered out and washed withmethanol and water. The filtrate was combined with washings, followed byconcentration under reduced pressure. The resulting residue wassubjected to gel filtration (Sephadex LH-20, eluent: 1:1 ethanol:water)to yield compound (15) (29 mg, quantitative).

Physical property data 1. Optical rotation: [α]_(D) -13.68° (C=0.833,water:ethanol=3:1) 2. Elemental analysis (for C₂₉ H₅₀ N₂ O₂₁)Calculated: C, 45.67%; H, 6.61%; N, 3.67% Found: C, 45.68%; H, 6.72%; N,3.56%

[EXAMPLE 2]

Production of Le^(a) and sialyl Lea-type sugar chain derivatives

(1) Preparation ofO-(2,3,4,6-tetra-O-benzoyl-β-D-galactopyranosyl)-(1→3)-2-O-acetyl-4,6-O-benzylidene-N-benzyloxycarbonyl-1,5-dideoxy-1,5-imino-D-glucitol

2-O-acetyl-4,6-O-benzylidene-N-benzyloxycarbonyl-1,5-dideoxy-1,5-imino-D-glucitol(1 g) and methyl-2,3,4,6-tetra-O-benzoyl-1-thio-β-D-galactopyranoside(2.9 g, 2 equivalents) were dissolved in dichloromethane (30 ml). Tothis solution, Molecular Sieves 4A (4 g), a desiccant, was added,followed by stirring at room temperature for 6 hours. After cooling themixture to -20° C., N-iodosuccinimide (2.11 g, 4.0 equivalents) andtrifluoromethanesulfonic acid (83 gl, 0.4 equivalents) were added,followed by overnight stirring at -20° C. to room temperature. Aftercompletion of the reaction was confmned by TLC, the Molecular Sieves wasfiltered out and then the filtrate was extracted with dichloromethaneand washed with sodium carbonate, sodium sulfite and water. The extractlayer was added over sodium sulfate and filtered. The filtrate wascombined with washings, followed by concentration under reducedpressure. The resulting residue was subjected to column chromatography(Wako Gel C-200, eluent: 1:4 ethyl acetate:hexane) to yield compound (1)(2.35 g, quantitative).

Physical property data 1. Optical rotation: [α]_(D) +19.85° (C=0.977,dichloromethane) 2. Elemental analysis (for C₅₇ H₅₁ NO₁₆) Calculated: C,68.05%; H, 5.11%; N, 1.39% Found: C, 67.96%; H, 5.21%; N, 1.31%

(2) Preparation ofO-(2,3,4,6-tetra-O-benzoyl-β-D-galactopyranosyl)-(1→3)-2-O-acetyl-6-O-benzyl-N-benzyloxycarbonyl-1,5-dideoxy-1,5-imino-D-glucitol

Compound (1) (2.35 g) was dissolved in tetrahydrofuran (50 ml). To thissolution, Molecular Sieves 3A (4 g), a desiccant, was added, followed bystirring at room temperature for 2 hours. Next, sodium cyanoborohydride(2.5 g, 15 equivalents <) was added for activation, after whichhydrochloric acid/ether was added drop by drop until generation ofgaseous hydrogen from the reaction mixture stopped, followed by stirringat room temperature for 1 hour. After completion of the reaction wasconfirmed by TLC, triethylamine was added at 0° C. to neutralize themixture. The Molecular Sieves was then filtered out and thoroughlywashed with dichloromethane and methanol. The filtrate was combined withwashings, followed by concentration under reduced pressure. Theresulting residue was extracted with dichloromethane and washed withwater. The extract layer was dried over sodium sulfate, filtered, andconcentrated under reduced pressure. The resulting residue was subjectedto column chromatography (Wako Gel C-200, eluent: 1:2 ethylacetate:hexane) to yield compound (2) (2.11 g, 90%).

Physical property data 1. Optical rotation: [α]_(D) +62.31° (C=1.133,dichloromethane) 2. Elemental analysis (for C₅₇ H₅₃ NO₁₆) Calculated: C,67.92%; H, 5.30%; N, 1.39% Found: C, 68.02%; H, 5.57%; N, 1.41%

(3) Preparation ofO-(2,3,4,6-tetra-O-benzoyl-β-D-galactopyranosyl)-(1→3)-O-[(2,3,4-tri-O-benzyl-α-L-fucopyranosyl)-(1→4)]-2-O-acetyl-6-O-benzyl-N-benzyloxycarbonyl-1,5-dideoxy-1,5-imino-D-glucitol

Compound (2) (500 mg) and methyl-2,3,4-tri-O-benzyl-1-thio-β-L-fucopyranoside (346 mg, 1.5 equivalents) were dissolved inbenzene (10 ml). To this solution, Molecular Sieves 4A (1 g), adesiccant, was added, followed by overnight stirring at roomtemperature. After cooling the mixture to 7° C.,dimethyl(methylthio)sulfonium triflate (682 mg, 4 equivalents) wasadded, followed by stirring at 7° C. to room temperature for 3.5 hours.After completion of the reaction was confirmed by TLC, methanol (5 ml)was added at 0° C. The mixture was neutralized with triethylamine, theMolecular Sieves was filtered out. The filtrate was combined withwashings, followed by concentration under reduced pressure. Theresulting residue was extracted with dichloromethane and the extract waswashed with water. The extract layer was dried over sodium sulfate,filtered, and concentrated under reduced pressure. The resulting residuewas subjected to column chromatography (Wako Gel C-200, eluent: 1:3ethyl acetate:hexane) to yield compound (3) (707 mg, quantitative).

Physical property data 1. Optical rotation: [α]_(D) -24.50° (C=1.020,dichloromethane) 2. Elemental analysis (for C₈₄ H₈₁ NO₂₀) Calculated: C,70.82%; H, 5.73%; N, 0.98% Found: C, 70.83%; H, 5.74%; N, 1.12%

(4) Preparation ofO-(2,3,4,6-tetra-O-benzoyl-β-D-galactopyranosyl)-(1→3)-O-[(.alpha.-L-fucopyranosyl)-(1→4)]-2-O-acetyl-1,5-dideoxy-1,5-imino-D-glucitol

Compound (3) (100 mg) was dissolved in methanol (13 ml) and formic acid(3 ml). To this solution, palladium black (100 mg), previouslycatalytically hydrogenated and washed with methanol, was added, followedby catalytic hydrogenation at room temperature. After completion of thereaction was confirmed by TLC, the palladium was filtered out and washedwith methanol. The filtrate was combined with washings, followed byconcentration under reduced pressure. The resulting residue wassubjected to column chromatography (Wako Gel C-200, eluents: a) 25:1dichloromethane: methanol, b) 20:1 dichloromethane:methanol) to yieldcompound (4) (65 mg, quantitative).

Physical property data 1. Optical rotation: [α]_(D) -13.02° (C=1.566,methanol) 2. Elemental analysis (for C₄₈ H₅₁ NO₁₈) Calculated: C,62.00%; H, 5.53%; N, 1.51% Found: C, 61.93%; H, 5.74%; N, 1.77%

(5) Preparation ofO-(β-D-galactopyranosyl)-(1→3)-O-[(α-L-fucopyranosyl)-(1→4)]-1,5-dideoxy-1,5-imino-D-glucitol

Compound (4) (47 mg) was dissolved in methanol (10 ml). To thissolution, sodium methoxide was added until the pH became almost 12,followed by overnight stirring at room temperature. After completion ofthe reaction was confirmed by TLC, the mixture was neutralized with ionexchange resin Amberlite IR-120 (H⁺). The resin was filtered out andwashed with methanol and water. The filtrate was combined with washings,followed by concentration under reduced pressure. The resulting residuewas subjected to gel filtration (Sephadex LH-20, eluent: 2:1ethanol:water) to yield compound (5) (23 mg, quantitative).

Physical property data 1. Optical rotation: [α]_(D) +1.01° (C=0.990,water:ethanol=2:1) 2. Elemental analysis (for C₁₈ H₃₃ NO₁₃) Calculated:C, 45.86%; H, 7.06%; N, 2.97% Found: C, 45.70%; H, 7.00%; N, 2.74%

(6) Preparation of O-(methyl5-acetamido-4,7,8,9-tetra-O-acetyl-3,5-dideoxy-D-glycero-α-D-galacto-2-nonulopyranosylonate)-(2→3)-O-(2,4,6-tri-O-benzoyl-β-D-galactopyranosyl)-(1→3)-2-O-acetyl-4,6-O-benzylidene-N-benzyloxycarbonyl-1,5-dideoxy-1,5-imino-D-glucitol

2-O-acetyl-4,6-O-benzylidene-N-benzyloxycarbonyl- 1,5-dideoxy-1,5-imino-D-glucitol (200 mg) and compound B (700 mg, 1.5 equivalents)were dissolved in dichloromethane (15 ml). To this solution, MolecularSieves 4A (1 g), a desiccant, was added, followed by overnight stirringat room temperature. After cooling the mixture to -20° C.,N-iodosuccinimide (316 mg, 3 equivalents) and trifluoromethanesulfonicacid (13 ml, 0.3 equivalents) were added, followed by overnight stirringat -20° C. to room temperature. After completion of the reaction wasconfirmed by TLC, the Molecular Sieves was filtered out, the filtratewas extracted with dichloromethane and washed with sodium carbonate,sodium thiosulfate and water. The extract layer was dried over sodiumsulfate and filtered. The filtrate was combined with washings, followedby concentration under reduced pressure. The resulting residue wassubjected to column chromatography (Merck Kiesel Gel 60, eluent: 250:4dichloromethane:methanol) to yield compound (6) (580 mg, 90%).

Physical property data 1. Optical rotation: [α]_(D) +8.81° (C=0.976,trichloromethane) 2. Elemental analysis (for C₇₀ H₇₄ N₂ O₂₇) Calculated:C, 61.13%; H, 5.42%; N, 2.04% Found: C, 60.87%; H, 5.23%; N, 1.89%

(7) Preparation of O-(methyl5-acetamido-4,7,8,9-tetra-O-acetyl-3,5-dideoxy-D-glycero-α-D-galacto-2-nonulopyranosylonate)-(2→3)-O-(2,4,6-tri-O-benzoyl-β-D-galactopyranosyl)-(1→3)-2-O-acetyl-6-O-benzyl-N-benzyloxycarbonyl-1,5-dideoxy-1,5-imino-D-glucitol

Compound (6) (200 ml) was dissolved in tetrahydrofuran (30 ml). To thissolution, Molecular Sieves 3A (400 mg), a desiccant, was added, followedby stirring at room temperature for 4 hours. Next, sodiumcyanoborohydride (150 mg, 15 equivalents <) was added for activation,after which hydrochloric acid/ether was added drop by drop at 0° C.until generation of gaseous hydrogen from the reaction mixture stopped,followed by stirring at 0° C. for 1.5 hours. After completion of thereaction was confirmed by TLC, the mixture was neutralized withtriethylamine. The Molecular Sieves was filtered out and thoroughlywashed with dichloromethane and methanol. The filtrate was combined withwashings, followed by concentration under reduced pressure. Theresulting residue was extracted with dichloromethane. The extract layerwas washed with water, followed by drying over sodium sulfate, filteredand concentrated under reduced pressure. The resulting residue wassubjected to column chromatography (Merck Kiesel Gel 60, eluent: 4:1ethyl acetate:hexane) to yield compound (7) (200 mg, quantitative).

Physical property data 1. Optical rotation: [α]_(D) +26.08° (C=0.920,dichloromethane) 2. Elemental analysis (for C₇₀ H₇₆ N₂ O₂₇) Calculated:C, 61.04%; H, 5.56%; N, 2.03% Found: C, 61.04%; H, 5.42%; N, 1.91%

(8) Preparation of O-(methyl5-acetamido-4,7,8,9-tetra-O-acetyl-3,5-dideoxy-D-glycero-α-D-galacto-2-nonulopyranosylonate)-(2→3)-O-(2,4,6-tri-O-benzoyl-β-D-galactopyranosyl)-(1→3)-O-[(2,3,4-tri-O-benzyl-α-L-fucopyranosyl)-(1→4)]-2-O-acetyl-6-O-benzyl-N-benzyloxycarbonyl-1,5-dideoxy-1,5-imino-D-glucitol

Compound (7) (200 mg) and compound A (101 mg, 1.5 equivalents) weredissolved in benzene (20 ml). To this solution, Molecular Sieves 4A (500mg), a desiccant, was added, followed by overnight stirring at roomtemperature. After cooling the mixture to 7° C.,dimethyl(methylthio)sulfonium triflate (200 mg, 4 equivalents) wasadded, followed by stirring at 7° C. to room temperature for 4 hours.After completion of the reaction was confirmed by TLC, methanol (10 ml)was added at 0° C. The mixture was neutralized with triethylamine. Then,the Molecular Sieves was filtered out. The filtrate was combined withwashings, followed by concentration under reduced pressure. Theresulting residue was extracted with dichloromethane. The extract layerwas washed with water, and dried over sodium sulfate, filtered, andconcentrated under reduced pressure. The resulting residue was subjectedto column chromatography (Wako Gel C-200, eluent: 2:1 ethylacetate:hexane) to yield compound (8) (230 mg, 89%).

Physical property data 1. Optical rotation: [α]_(D) -16.49° (C=0.970,dichloromethane) 2. Elemental analysis (for C₉₇ H₁₀₄ N₂ O₃₁) Calculated:C, 64.95%; H, 5.84%; N, 1.56% Found: C, 64.70%; H, 5.86%; N, 1.36%

(9) Preparation of O-(methyl5-acetamido-4,7,8,9-tetra-O-acetyl-3,5-dideoxy-D-glycero-α-D-galacto-2-nonulopyranosylonate)-(2→3)-O-(2,4,6-tri-O-benzoyl-β-D-galactopyranosyl)-(1→3)-O-[(α-L-fucopyranosyl)-(1→4)]-2-O-acetyl-1,5-dideoxy-1,5-imino-D-glucitol

Compound (8) (60 mg) was dissolved in methanol (10 ml) and formic acid(10 ml). To this solution, palladium black (60 mg), previouslycatalytically hydrogenated and washed with methanol, was added, followedby catalytic hydrogenation at room temperature. After completion of thereaction was confirmed by TLC, the palladium was filtered out and washedwith methanol. The filtrate was combined with washings, followed byconcentration under reduced pressure. The resulting residue wassubjected to column chromatography (Wako Gel C-200, eluents: a) 25:1dichloromethane:methanol, b) 20:1 dichloromethane:methanol) to yieldcompound (9) (43 mg, quantitative) from eluent b).

Physical property data 1. Optical rotation: [α]_(D) -25.80° (C=1.350,methanol) 2. Elemental analysis (for C₆₁ H₇₄ N₂ O₂₉) Calculated: C,56.39%; H, 5.74%; N, 2.16% Found: C, 56.40%; H, 5.68%; N, 2.40%

(10) Preparation ofO-(5-acetamido-3,5-dideoxy-D-glycero-α-D-galacto-2-nonulopyranosylonicacid)-(2→3)-O-(β-D-galactopyranosyl)-(1→3)-O-[(α-L-fucopyranosyl)-(1→4)]-1,5-dideoxy-1,5-imino-D-glucitol

Compound (9) (40 mg) was dissolved in methanol (10 ml). To thissolution, sodium methoxide was added until the pH became almost 12,followed by overnight stirring at room temperature. Then, a 0.2N aqueoussolution of potassium hydroxide (5 ml) was added, followed by overnightstirring at room temperature. After completion of the reaction wasconfirmed by TLC, the mixture was neutralized with ion exchange resinAmberlite IR-120 (H⁺). The resin was filtered out and thoroughly washedwith methanol and water. The filtrate was combined with washings,followed by concentration under reduced pressure. The resulting residuewas subjected to gel filtration (Sephadex LH-20, eluent: 1:1ethanol:water) to yield compound (10) (23 mg, quantitative).

Physical property data 1. Optical rotation: [α]_(D) -4.03° (C=0.743,water:ethanol=3:1) 2. Elemental analysis (for C₂₉ H₅₀ N₂ O₂₁)Calculated: C, 45.67%; H, 6.61%; N, 3.67% Found: C, 45.86%; H, 6.84%; N,3.65%

[EXAMPLE 3]

Preparation of glucosamine-type sialyl Le^(x) derivatives

(1) Preparation of4,6-O-benzylidene-N-benzyloxycarbonyl-3-O-chloroacetyl-1,5-dideoxy-1,5-imino-2-O-mesyl-D-glucitol

4,6-O-benzylidene-N-benzyloxycarbonyl-3-O-chloroacetyl-1,5-dideoxy-1,5-imino-D-glucitol (1.27 g) was dissolved indichloromethane (50 ml) and pyridine (5 ml). To this solution,methanesulfonyl chloride (0.38 ml, 1.8 equivalents) was added at -20°C., followed by stirring at -20° C. to 0° C. for 8 hours. Aftercompletion of the reaction was confirmed by TLC, the mixture wasextracted with dichloromethane and washed with 2N hydrochloric acid andwater. The extract layer was dried over sodium sulfate and filtered. Thefiltrate was combined with washings, followed by concentration underreduced pressure to yield compound (1) (1.49 g, quantitative).

Physical property dam 1. Optical rotation: [α]_(D) -16.28° (C=1.253,trichloromethane) 2. Elemental analysis (for C₂₄ H₂₆ N₂ O₉ SCl)Calculated: C, 53.38%; H, 4.85%; N, 2.59% Found: C, 53.40%; H, 4.62%; N,2.87%

(2) Preparation of2,3-anhydro-4,6-O-benzylidene-N-benzyloxycarbonyl-1,5-dideoxy-1,5-imino-D-mannitol

Compound (1) (1.4 g) was dissolved in 1,4-dioxane (5 ml) and methanol(15 ml). After cooling the solution to 0° C., sodium methoxide (0.89 ml,6 equivalents) was added, followed by stirring at 0° C. for 5 minutes.After completion of the reaction was confirmed by TLC, the mixture wasconcentrated under reduced pressure at 20° C. The resulting residue wasextracted with dichloromethane. The extract layer was washed with waterfollowed by drying over sodium sulfate and filtered. The filtrate wascombined with washings, followed by concentration under reduced pressureat 20° C. The resulting residue was subjected to column chromatography(Wako Gel C-200, eluent: 400:1 dichloromethane:methanol) to yieldcompound (2) (0.95 g, quantitative).

Physical property data 1. Optical rotation: [α]_(D) +46.65° (C=0.853,dichloromethane) 2. Elemental analysis (for C₂₁ H₂₁ NO₅) Calculated: C,68.65%; H, 5.76%; N, 3.81% Found: C, 68.64%; H, 5.90%; N, 3.61%

(3) Preparation of 2-azide-4,6-O-benzylidene-N-benzyloxycarbonyl-1,2,5-trideoxy-1,5 -imino-D-glucitol

Compound (2) (3.71 g) was dissolved in N,N-dimethylformamide (15 ml). Tothis solution, sodium azide (6.56 g, 10 equivalents ) was added,followed by overnight stirring at 110° C. After completion of thereaction was confirmed by TLC, the sodium azide was filtered out,followed by concentration under reduced pressure. The resulting residuewas extracted with dichloromethane. The extract layer was washed withwater, dried over sodium sulfate and filtered. The filtrate was combinedwith washings, followed by concentration under reduced pressure. Theresulting residue was subjected to column chromatography (Wako GelC-200, eluent: 1:3 ethyl acetate:hexane) to yield compound (3) (1.66 g,40%).

Physical property data 1. Optical rotation: [α]_(D) -15.95° (C=1.216,dichloromethane) 2. Elemental analysis (for C₂₁ H₂₂ N₄ O₅) Calculated:C, 61.46%; H, 5.40%; N, 13.65% Found: C, 61.56%; H, 5.29%; N, 13.70%

(4) Preparation of2-acetamido-4,6-O-benzylidene-N-benzyloxycarbonyl-1,2,5-trideoxy-1,5-imino-D-glucitol

Compound (3) (280 mg) was dissolved in 1,2-dichloromethane (10 ml).After heating the solution to 45° C., triphenylphosphine (385 mg, 2equivalents) was added, followed by stirring at 45° C. for 30 minutes.Next, water (0.15 ml, 10 equivalents <) was added, followed by overnightstirring at 45° C. After completion of the reaction was confirmed byTIE, the mixture was concentrated under reduced pressure. The resultingresidue was dissolved in methanol (10 ml). To this solution, aceticanhydride (71 μl, 1.1 equivalents) was added, followed by stirring atroom temperature for 4 hours. After completion of the reaction wasconfirmed, pyridine (2 ml) was added at 0° C., followed by concentrationunder reduced pressure. The resulting residue was extracted withdichloromethane and washed with 2N hydrochloric acid and water. Theextract layer was dried over sodium sulfate and filtered. The filtratewas combined with washings, followed by concentration under reducedpressure. The resulting residue was subjected to column chromatography(Wako Gel C-200, eluents: a) 80:1 dichloromethane:methanol, b) 50:1dichloromethane:methanol) to yield compound (4) (270 mg, 93%) fromeluent b).

Physical property data 1. Optical rotation: [α]_(D) +3.22° (C=0.743,dichloromethane) 2. Elemental analysis (for C₂₃ H₂₆ N₂ O₆) Calculated:C, 64.78%; H, 6.15%; N, 6.57% Found: C, 64.51%; H, 6.36%; N, 6.39%

(5) Preparation ofO-(2,3,4-tri-O-benzyl-α-L-fucopyranosyl)-(1→3)-2-acetamido-4,6-O-benzylidene-N-benzyloxycarbonyl-1,2,5-trideoxy-1,5-imino-D-glucitol

Compound (4) (250 mg) andmethyl-2,3,4-tri-O-benzyl-1-thio-β-L-fucopyranoside (compound A) (372mg, 1.2 equivalents) were dissolved in benzene (20 ml). To thissolution, Molecular Sieves 4A (700 mg), a desiccant, was added, followedby overnight stirring at room temperature. Next,dimethyl(methylthio)sulfonium triflate (608 mg, 4.0 equivalents) wasadded thereto at 7° C., followed by stirring at 7° C. to roomtemperature for 3 hours. After completion of the reaction was confirmedby TLC, methanol (10 ml) was added at 0° C. The mixture was neutralizedwith triethylamine and filtered to separate the Molecular Sieves. Thefiltrate was then combined with washings, followed by concentrationunder reduced pressure. The resulting residue was extracted withdichloromethane. The extract layer washed with water, dried over sodiumsulfate and filtered, followed by concentration under reduced pressure.The resulting residue was subjected to column chromatography (Wako GelC-200, eluent: 1:2 ethyl acetate:hexane) to yield compound (5) (494 mg,quantitative).

Physical property data 1. Optical rotation: [α]_(D) -83.24° (C=0.973,dichloromethane) 2. Elemental analysis (for C₅₀ H₅₄ N₂ O₁₀) Calculated:C, 71.24%; H, 6.46%; N, 3.32% Found: C, 71.31%; H, 6.36%; N, 3.61%

(6) Preparation ofO-(2,3,4-tri-O-benzyl-α-fucopyranosyl)-(1→3)-2-acetamido-6-O-benzyl-N-benzyloxycarbonyl-1,2,5-trideoxy-1,5-imino-D-glucitol

Compound (5) (494 mg) was dissolved in tetrahydrofuran (30 ml). To thissolution, Molecular Sieves 3A (1 g), a desiccant, was added, followed bystirring at room temperature for 3 hours. Next, sodium cyanoborohydride(600 mg, 15 equivalents <) was added for activation, after whichhydrochloric acid/ether was added drop by drop until generation ofgaseous hydrogen from the reaction mixture stopped, followed by stirringat room temperature for 4 hours. After completion of the reaction wasconfirmed by TLC, triethylamine was added at 0° C. to neutralize themixture. The Molecular Sieves was filtered out and thoroughly washedwith dichloromethane and methanol. The filtrate was combined withwashings, followed by concentration under reduced pressure. Theresulting residue was extracted with dichloromethane. The extract layerwas washed with water, dried over sodium sulfate and filtered. Thefiltrate was concentrated under reduced pressure, and the resultingresidue was subjected to column chromatography (Wako Gel C-200, eluent:1:1 ethyl acetate:hexane) to yield compound (6) (495 mg, quantitative).

Physical property dam 1. Optical rotation: [α]_(D) -15.39° (C=0.926,dichloromethane) 2. Elemental analysis (for C₅₀ H₅₆ N₂ O₁₀) Calculated:C, 71.07%; H, 6.68%; N, 3.32% Found: C, 71.28%; H, 6.81%; N, 3.28%

(7) Preparation of O-(methyl5-acetamido-4,7,8,9-tetra-O-acetyl-3,5-dideoxy-D-glycero-α-D-galacto-2-nonulopyranosylonate)-(2→3)-O-(2,4,6-tri-O-benzoyl-β-D-galactopyranosyl)-(1→4)-O-[(2,3,4-tri-O-benzyl-α-L-fucopyranosyl)-(1→3)]-2-acetamido-6-O-benzyl-N-benzyloxycarbonyl-1,2,5-trideoxy-1,5-imino-D-glucitol

Compound (6) (130 mg) and compound B (230 mg, 1.5 equivalents) weredissolved in dichloromethane (20 ml). To this solution, Molecular Sieves4A (400 mg), a desiccant, was added, followed by overnight stirring atroom temperature. After cooling the mixture to -20° C.,N-iodosuccinimide (110 mg, 3 equivalents) and trifluoromethanesulfonicacid (5 μl, 0.3 equivalents) were added, followed by overnight stirringat -20° C. to room temperature. After completion of the reaction wasconfirmed by TLC, the Molecular Sieves was filtered out. The reactionmixture was extracted with dichloromethane. The extract layer was washedwith sodium carbonate, sodium sulfite and water, followed by drying oversodium surf ate and filtered. The filtrate was combined with washings,followed by concentration under reduced pressure. The resulting residuewas subjected to column chromatography (Merck Kiesel Gel 60, eluent: 2:1ethyl acetate:hexane) to yield compound (7) (111 mg, 40%).

Physical property data 1. Optical rotation: [α]_(D) -6.91° (C=1.214,dichloromethane) 2. Elemental analysis (for C₉₇ H₁₀₅ N₃ O₃₀) Calculated:C, 64.98%; H, 5.90%; N, 2.34% Found: C, 65.11%; H, 6.18%; N, 2.35%

(8) Preparation of O-(methyl5-acetamido-4,7,8,9-tetra-O-acetyl-3,5-dideoxy-D-glycero-α-D-galacto-2-nonulopyranosylonate)-(2→3)-O-(2,4,6-tri-O-benzoyl-β-D-galactopyranosyl)-(1→4)-O-[(α-L-fucopyranosyl)-(1→3)]-2-acetamido-1,2,5-trideoxy-N-methyl-1,5-imino-D-glucitol

Compound (7) (66 mg) was dissolved in methanol (10 ml) and formic acid(10 ml). To this solution, palladium black (66 mg), previouslycatalytically hydrogenated and washed with methanol, was added, followedby catalytic hydrogenation at room temperature for 10 days. Aftercompletion of the reaction was confirmed by TLC, the palladium wasfiltered out and washed with methanol. The filtrate was combined withwashings, followed by concentration under reduced pressure. Theresulting residue was subjected to column chromatography (Wako GelC-200, eluent: 15:1 dichloromethane:methanol) to yield compound (8) (48mg, quantitative).

Physical property data 1. Optical rotation: [α]_(D) -19.20° (C=1.583,methanol) 2. Elemental analysis (for C₆₂ H₇₇ N₃ O₂₈) Calculated: C,56.75%; H, 5.91%; N, 3.20% Found: C, 56.61%; H, 6.08%; N, 3.02%

(9) Preparation ofO-(5-acetamido-3,5-dideoxy-D-glycero-α-D-galacto-2-nonulopyranosylonicacid)-(2→3)-O-(β-D-galactopyranosyl)-(1→4)-O-[(α-L-fucopyranosyl)-(1→3)]-2-acetamido-1,2,5-trideoxy-N-methyl-1,5-imino-D-glucitol

Compound (8) (44 mg) was dissolved in methanol (10 ml). To thissolution, sodium methoxide was added until the pH became almost 12,followed by overnight stirring at room temperature. Then, a 0.2N aqueoussolution of potassium hydroxide (5 ml) was added, followed by overnightstirring at room temperature. After completion of the reaction wasconfirmed by TLC, the solution was neutralized with ion exchange resinAmberlite IR-120 (H⁺). The resin was filtered out and thoroughly washedwith methanol and water. The filtrate was combined with washings,followed by concentration under reduced pressure. The resulting residuewas subjected to gel filtration (Sephadex LH-20, eluent: 1:3ethanol:water) to yield compound (9) (29 mg, quantitative).

Physical property data 1. Optical rotation: [α]_(D) -3.93° (C=0.966,water:ethanol=4:1) 2. Elemental analysis (for C₃₂ H₅₅ N₃ O₂₁)Calculated: C, 47.00%; H, 6.78%; N, 5.14% Found: C, 46.97%; H, 6.48%; N,5.06%

(10) Preparation of O-(methyl5-acetamido-4,7,8,9-tetra-O-acetyl-3,5-dideoxy-D-glycero-α-D-galacto-2-nonulopyranosylonate)-(2→3)-O-(2,4,6-tri-O-benzoyl-β-D-galactopyranosyl)-(1→3)-2-acetamido-4,6-O-benzylidene-N-benzyloxycarbonyl-1,2,5-trideoxy-1,5-imino-D-glucitol

Compound (4) (150 mg) and compound B (525 mg, 1.5 equivalents) weredissolved in dichloromethane (15 ml). To this solution, Molecular Sieves4A (800 mg), a desiccant, was added, followed by stirring at roomtemperature for 5 hours. After cooling the mixture to -20° C.,N-iodosuccinimide (237 mg, 3 equivalents) and trifluoromethanesulfonicacid (10 μl, 0.3 equivalents) were added, followed by overnight stirringat -20° C. to room temperature. After completion of the reaction wasconfirmed by TLC, the Molecular Sieves was filtered out. The filtratewas extracted with dichloromethane and washed with sodium carbonate,sodium sulfite and water. The extract layer was dried over sodium surfate and faltered. The filtrate was combined with washings, followed byconcentration under reduced pressure. The resulting residue wassubjected to column chromatography (Merck Kiesel Gel 60, eluents: a) 3:1ethyl acetate:hexane, b) 4:1 ethyl acetate:hexane) to yield compound(10) (220 mg, 46%).

Physical property data 1. Optical rotation: [α]_(D) -0.24° (C=0.816,dichloromethane) 2. Elemental analysis (for C₇₀ H₇₅ N₃ O₂₆) Calculated:C, 61.18%; H, 5.50%; N, 3.06% Found: C, 61.11%; H, 5.69%; N, 3.21%

(11 ) Preparation of O-(methyl5-acetamido-4,7,8,9-tetra-O-acetyl-3,5-dideoxy-D-glycero-α-D-galacto-2-nonulopyranosylonate)-(2→3)-O-(2,4,6-tri-O-benzoyl-β-D-galactopyranosyl)-(1→3)-2-acetamido-6-O-benzyl-N-benzyloxycarbonyl-1,2,5-trideoxy-1,5-imino-D-glucitol

Compound (10) (190 mg) was dissolved in tetrahydrofuran (30 ml). To thissolution, Molecular Sieves 3A (400 mg), a desiccant, was added, followedby stirring at room temperature for 5 hours. Next, sodiumcyanoborohydride (170 mg, 15 equivalents <) was added for activation,followed by stirring at 0° C. for 4.5 hours. After completion of thereaction was confirmed by TLC, the mixture was neutralized withtriethylamine. The Molecular Sieves was filtered out and thoroughlywashed with dichloromethane and methanol. The filtrate was combined withwashings, followed by concentration under reduced pressure. Theresulting residue was extracted with dichloromethane. The extract layerwas washed with water, and dried over sodium sulfate and filtered. Thetiltrate was concentrated under reduced pressure. The resulting residuewas subjected to column chromatography (Wako Gel C-200, eluent: 4:1ethyl acetate:hexane) to yield compound (11 ) (190 mg, quantitative).

Physical property data 1. Optical rotation: [α]_(D) +16.04° (C=0.723,dichloromethane) 2. Elemental analysis (for C₇₀ H₇₇ N₃ O₂₆) Calculated:C, 61.09%; H, 5.64%; N, 3.05% Found: C, 60.82%; H, 5.55%; N, 3.11%

(12) Preparation of O-(methyl5-acetamido-4,7,8,9-tetra-O-acetyl-3,5-dideoxy-D-glycero-α-D-galacto-2-nonulopyranosylonate)-(2→3)-O-(2,4,6-tri-O-benzoyl-β-D-galactopyranosyl)-(1→3)-O-[(2,3,4-tri-O-benzyl-α-L-fucopyranosyl)-(1→4)]-2-acetamido-6-O-benzyl-N-benzyloxycarbonyl-1,2,5-trideoxy-1,5-imino-D-glucitol

Compound (11) (70 mg) and compound A (35 mg, 1.5 equivalents) weredissolved in benzene (15 ml). To this solution, Molecular Sieves 4A (200mg), a desiccant, was added, followed by stirring overnight at roomtemperature. After cooling the mixture to 7° C., N-iodosuccinimide (35mg, 3 equivalents) and trifluoromethanesulfonic acid (1.5 μl, 0.3equivalents) were added thereto, followed by stirring at 7° C. to roomtemperature for 5 hours. After completion of the reaction was confirmedby TLC, the Molecular Sieves was filtered out. The filtrate wasextracted with dichloromethane and the extract was washed with sodiumcarbonate, sodium sulfite and water. The extract layer was dried oversodium sulfate and filtered. The filtrate was combined with washings,followed by concentration under reduced pressure. The resulting residuewas subjected to column chromatography (Wako Gel C-200, eluent: 3:1ethyl acetate:hexane) to yield compound (12) (50 mg, 55%).

Physical property data 1. Optical rotation: [α]_(D) -18.74° (C=1.462,dichloromethane) 2. Elemental analysis (for C₉₇ H₁₀₅ N₃ O₃₀) Calculated:C, 64.98%; H, 5.90%; N, 2.34% Found: C, 65.13%; H, 6.01%; N, 2.62%

(13) Preparation ofO-(methyl-5-acetamido-4,7,8,9-tetra-O-acetyl-3,5-dideoxy-D-glycero-α-D-galacto-2-nonulopyranosylonate)-(2→3)-O-(2,4,6-tri-O-benzoyl-.beta.-D-galactopyranosyl)-(1→3)-O-[(α-L-fucopyranosyl)-(1→4)]-2-acetamido-1,2,5-trideoxy-N-methyl-1,5-imino-D-glucitol

Compound (12) (61 mg) was dissolved in methanol (10 ml) and formic acid(10 ml). To this solution, palladium black (60 mg), previouslycatalytically hydrogenated and washed with methanol, was added, followedby catalytic hydrogenation at room temperature while stirring for 7days. After completion of the reaction was confirmed by TLC, thepalladium was filtered out and washed with methanol. The filtrate wascombined with washings, followed by concentration under reducedpressure. The resulting residue was subjected to column chromatography(Wmko Gel C-200, eluents: a) acetone, b) methanol) to yield compound(13) (44 mg, quantitative).

Physical property data 1. Optical rotation: [α]_(D) -9.18° (C=1.481,methanol) 2. Elemental analysis (for C₆₂ H₇₇ N₃ O₂₈) Calculated: C,56.75%; H, 5.91%; N, 3.20% Found: C, 56.50%; H, 5.87%; N, 3.49%

(14) Preparation ofO-(5-acetamido-3,5-dideoxy-D-glycero-α-D-galacto-2-nonulopyranosylonicacid)-(2→3)-O-(β-D-galactopyranosyl)-(1→3)-O-[(α-L-fucopyranosyl)-(1→4)]-2-acetamido-1,2,5-trideoxy-N-methyl-1,5-imino-D-glucitol

Compound (13) (30 mg) was dissolved in methanol (10 ml). To thissolution, sodium methoxide was added until the pH became almost 12,followed by overnight stirring at room temperature. Then, a 0.2N aqueoussolution of potassium hydroxide (6 ml) was added, followed by stirringat room temperature for 2 days. After completion of the reaction wasconfirmed by TLC, the mixture was neutralized with ion exchange resinAmberlite IR-120 (H⁺). The resin was filtered out and thoroughly washedwith methanol and water. The tiltrate was combined with washings,followed by concentration under reduced pressure. The resulting residuewas subjected to gel filtration (Sephadex LH-20, eluent: 1:3ethanol:water) to yield compound (14) (19 mg, quantitative).

Physical property data 1. Optical rotation: [α]_(D) -23.47° (C=0.230,water:ethanol=4:1) 2. Elemental analysis (for C₃₂ H₅₅ N₃ O₂₁)Calculated: C, 47.00%; H, 6.78%; N, 5.14% Found: C, 46.80%; H, 6.50%; N,5.16%

[EXAMPLE 4]

Preparation of N-methyl Le^(a) -type sugar chain derivatives

The same procedure as above was followed to yieldO-(β-D-galactopyranosyl)-(1→3)-O-[(α-L-fucopyranosyl)-(1→4)]-1,5-dideoxy-N-methyl-1,5-imino-D-glucitol.

Physical property data 1. Optical rotation: [α]_(D) +1° (C=0.500,water:ethanol=2:1 ) 2. Elemental analysis (for C₁₉ H₃₅ NO₁₃) Calculated:C, 47.01%; H, 7.27%; N, 2.89% Found: C, 47.02%; H, 7.38%; N, 2.78%

[EXAMPLE 5]

Preparation of N-methyl SLe^(a) -type sugar chain derivative

The same procedure as above was followed to yieldO-(5-acetamido-3,5-dideoxy-D-glycero-α-D-galacto-2-nonulopyranosylonicacid)-(2→3)-O-(β-D-galactopyranosyl)-(1→3)-O-[(α-L-fucopyranosyl)-(1→4)]-1,5-dideoxy-N-methyl-1,5-imino-D-glucitol.

Physical property data 1. Optical rotation: [α]_(D) -26° (C=0.500,water:ethanol=4:1) 2. Elemental analysis (for C₃₀ H₅₂ N₂ O₂₁)Calculated: C, 46.39%; H, 6.75%; N, 3.61% Found: C, 46.29%; H, 6.91%; N,3.62%

The present invention is hereinafter described in more detail by meansof the following test example.

[TEST EXAMPLE]

Effects of synthetic sugar chains on binding of activated human vascularendothelial cells and cultured human leukemia cell HL60

Cell culture

Cultured human vascular endothelial cells were isolated from theumbilical vein via collagenase treatment and cultured in a flask forculture, coated with 1% gelatin or at the concentration of 0.02 mg/cm²fibronectin. The growth medium used was prepared by adding 15% fetalcalf serum (FCS), 45 mg/1ECGS, 90 mg/1 heparin and 40 mg/l gentamycin to199 medium. Cultured human leukemia cell HL60 was grown and maintainedin RPMI1640 medium containing 10% FCS. For both types of cells, culturewas conducted in a CO₂ incubator (5% CO₂, 95% air) at 37° C.

Measurement of cell adhesion

Cultured human umbilical venous cells (HUVEC) were seeded over a 96-wellculture plate coated with 0.1% gelatin, and cultured on growth mediumuntil they became confluent. The cells were washed with Dulbecco'smodified Eagle medium (DMEM) containing 10% FCS, and cultured for 4hours in a 37° C. CO₂ incubator after adding 100 μl of 10% FCS DMEMcontaining recombinant human interleukin-1β (IL-1β) (activation). TheHL60 cells were twice washed with serum-free DMEM, suspended in Hanks'solution containing 0.5% glutaraldehyde, and fixed, with ice cooling,for 30 minutes. After fixation, the cells were twice washed withserum-free DMEM to remove the fixative. Then, the number of cells wasadjusted to 2×10⁶ cells/ml using DMEM containing 10% FCS, and stored inice until using. After activation, the HUVEC was washed with DMEMcontaining 10% FCS, and incubated at room temperature for 30 minutesafter adding 50 μl of 10% FCS DMEM containing either an anti-ELAM-1antibody (BBA 2, British Biotechnology Lid, Abinton) or a sample. Thefixed HL60 cells were added at 50 μl per well, followed by incubation atroom temperature for 45 minutes. After unbound cells were washed out,the central portion of each well was photographed. The adhesive cellsseen in the imaging field were counted. The effect obtained by theaddition of the sample was assessed as a percent ratio to the number ofHL60 cells bound to the IL-1β-activated HUVEC. The results are given inTables 1 and 2.

Table 1 shows that the SLe^(x) derivative of the compound (15) of theinvention, described in Example 1, and the Le^(x) derivative of compound(12), described in Example 1, suppressed the HL60 cell adhesion to theIL-1β-activated HUVEC by 29.9% and 37.6%, respectively, when they werepresent at a concentration of 100 μg/ml, demonstrating ELAM-1-dependentcell adhesion inhibitory activity.

Table 2 shows that the SLe^(x) derivative of the compound (9) of theinvention, described in Example 3, the SLe^(a) derivative of compound(14), described in Example 3, the Le^(a) derivative described in Example4, and the SLe^(a) derivative described in Example 5, suppressed theHL60 cell adhesion to IL-1β-activated HUVEC by 49.6%, 43.1%, 29.8% and71.1%, respectively, when they were present at a concentration of 100μg/ml, demonstrating ELAM-1-dependent cell adhesion inhibitory activity.

                  TABLE 1                                                         ______________________________________                                        Inhibitory action of SLe.sup.x and Le.sup.x -type sugar chain                 derivatives on ELAM-1-dependent adhesion of                                   cultured human leukemia cell HL60                                                       contentration                                                                            Number of Number of                                      Treatment (μg/ml) samples   adhering cells (%)                             ______________________________________                                        Basal                5         36.8   (28.1)                                  Control              5         131.0  (100.0)                                 α-ELAM                                                                            50         5         27.6   (21.1)                                  Example 1 (15)                                                                          10         5         117.8  (89.9)                                  Example 1 (15)                                                                          100        5         91.8   (70.1)                                  Example 1 (12)                                                                          10         5         120.0  (91.6)                                  Example 1 (12)                                                                          100        5         81.8   (62.4)                                  ______________________________________                                         Note:                                                                         "Basal" shows adhesion to nonactivated HUVEC;                                 "Control" shows adhesion to HUVEC activated with 10 U/ml IL1β;           "ELAM" shows adhesion to activated HUVEC in the case which antiELAM-1         antibody was added;                                                           "Example 1 (15)" shows adhesion to activated HUVEC in the case which an       SLe.sup.xtype substance, inventive compound (15) described in Example 1       was added;                                                                    "Example 1 (12)" shows adhesion to activated HUVEC in the case which an       Le.sup.xtype substance, compound (12) described in Example 1 was added.  

                  TABLE 2                                                         ______________________________________                                        Inhibitory action of SLe.sup.a, Le.sup.a and SLe.sup.x -type sugar chain      derivatives on ELAM-1-dependent adhesion of cultured                          human leukemia cell HL60                                                                contentration                                                                            Number of Number of                                      Treatment (μg/ml) samples   adhering cells (%)                             ______________________________________                                        Basal                5         28.2   (7.4)                                   Control              5         383.4  (100.0)                                 α-ELAM                                                                            25         5         75.0   (19.6)                                  Example 3 (9)                                                                           100        5         193.2  (50.4)                                  Example 3 (14)                                                                          100        5         218.2  (56.9)                                  Example 4 100        5         269.0  (70.2)                                  Example 5 100        5         110.8  (28.9)                                  ______________________________________                                         Note:                                                                         "Basal" shows adhesion to nonactivated HUVEC;                                 "Control" shows adhesion to HUVEC activated with 10 U/ml IL1β;           "ELAM" shows adhesion to activated HUVEC in the case which antiELAM-1         antibody was added;                                                           "Example 3 (9)" shows adhesion to activated HUVEC in the case that an         SLe.sup.xtype substance, inventive compound (9) described in Example 3 wa     added;                                                                        "Example 3 (14)" shows adhesion to activated HUVEC in the case which an       SLe.sup.atype substance of compound (14) described in Example 3 was added     "Example 4" shows adhesion to activated HUVEC in the case that an             Le.sup.atype substance, the compound described in Example 4 was added;        "Example 5" shows adhesion to activated HUVEC in the case that an             SLe.sup.atype substance, the compound described in Example 5 was added.  

When administered as a pharmaceutical, the compounds of the presentinvention can be administered as such or in a pharmaceutical compositioncomprising 0.1 to 99.5%, preferably 0.5 to 90% of the compound in apharmaceutically acceptable nontoxic inert carrier, to animals,including humans.

At least one solid, semi-solid or liquid diluents, filling agents, andother formulation aids are used as carriers. It is desirable that thepharmaceutical composition be administered in an administration unitform. The pharmaceutical composition according to the present inventioncan be administered orally, intratissularly, topically (percutaneousadministration etc.) or perfectally, in dosage forms suitable thereto.For example, intratissular administration is preferred.

It is desirable that the dose of the compound of the present invention,as an anti-inflammatory agent, be chosen in view of age, body weight andother factors of the patient, route of administration, nature andseverity of the target disease and other factors. The dose range isusually from 100 mg to 3 g/day, preferably from 500 mg to 1 g/day foreach adult, as the amount of the active ingredient of the presentinvention. As the case may be, the dose may be lower or higher. It isalso desirable that the daily dose be divided in one to three portions.

What is claimed is:
 1. A Lewis-type sugar chain derivative representedby the following formula: wherein R¹ represents hydrogen, a lower alkyl,a lower alkenyl or a lower alkynyl; R² and R³ differ from each other andrepresent a galactosyl, sialylgalactosyl or fucosyl group; and R⁴represents a hydroxyl group or an acetamido group: ##STR25##
 2. A LewisX derivative, according to claim 1, represented by the followingformula: ##STR26##
 3. A Lewis X derivative, according to claim 1,represented by the following formula: ##STR27##
 4. A Lewis X derivative,according to claim 1, represented by the following formula: ##STR28## 5.A Lewis A derivative, according to claim 1, represented by the followingformula: ##STR29##
 6. A Lewis A derivative, according to claim 1,represented by the following formula: ##STR30##
 7. A Lewis A derivative,according to claim 1, represented by the following formula: ##STR31## 8.A Lewis A derivative, according to claim 1, represented by the followingformula: ##STR32##
 9. A method for inhibiting cell adhesion in mammals,which comprises administering to a mammal in need thereof an effectiveamount of the compound of claim 1, or a pharmaceutically acceptable saltthereof or a solvate of said compound or said salt in combination with apharmaceutically acceptable carrier.
 10. A pharmaceutical compositionuseful for inhibiting cell adhesion in mammals, which comprises atherapeutically effective amount of the compound of claim 1, or apharmaceutically acceptable salt thereof or a solvate of said compoundor said salt in combination with a pharmaceutically acceptable carrier.